A generic Phosphatase and pyrophosphatase assay

Sunday, December 28, 2003

Description
This review discusses the EnzChek phosphatase assay kit comercially available from Molecular Probes Inc. that we have found to be very valuable to assay probable phosphatase activity of enzymes. The assay (done in-vitro, using a regular UV spectrophotometer) can be easily modified to probe any reaction that generates inorganic phosphate or pyrophosphate during catalysis.

Procedure
Rationale:
The method works by coupling any enzymatic reaction that generates inorganic phosphate to another enzyme, purine nucleoside phosphorylase (PNP). The method was originally developed by Webb in 1992. PNP uses MESG substrate (2-amino-6-mercapto-7-methylpurine riboside; abs max of 330 nm) and inorganic pi and converts it into ribose-1-phosphate and a product that has absorbtion max of 360 nm. (for further details see enzchek phosphatase product info for E6646 at probes.com).
As your enzyme releases phosphate in the cuvette, it is rapidly used up by this reaction resulting in a steady increase of UV absorbance at 360 nm. The reaction is relatively straightforward to do except that one has to be very careful of inadvertant phosphate contamination.

Generation of std curve: The kit comes with phosphate standard that canbe used to calibrate reaction rates over a wide range of phosphate concentrations.

Recipes
The Reaction:
To a 1 ml cuvette add the following:
1. 50 microL of reaction buffer (supplied as 20x)
2. 200 microL of MESG
3. 10 microL of PNP enzyme
4. x microL of your enzyme of interest (typically 1-5 micro L)
5. y microL of your substrate (less than 20 microL)
6. Finally 740 -x-y microL of Pure H2O (careful of water with phosphate contamination). preferably do a blank without enzyme or substrate and see for increase in UV.

Incubate for a short while (recommended 20 min) and measure absorbance at 360 nm on any standard UV spectrophotometer. Preferably do in triplicates with proper controls.

The kit is a bit expensive and works out to be about 2 dollars per reaction for 1ml volumes. you can also try reducing it to 500 microL reactions if you notice no loss in sensitivity.

Kinetic measurements:

One can perform rate dependent kinetic measurements by starting the reaction by either adding the substrate or the enzyme and immediately start monitoring increase in absorbance at 360nm. Keep monitoring till the increase is no longer a straight line with constant slope.

Supplies
Nothing in particular.,
a few tips, a few accurate pippettes 1-10 microL for substrates and 1 ml for H2O, a cuvette for monitoring in visible range, and a UV spetrophotometer.
Of course the EnzChek phosphatase or pyrophosphatase Kit.

Tips
While many enzymes have been studied using this method successfully (or so the product info states), we have been successful in performing this reaction with phosphoserine phosphatase (that releases inorganic Pi) and NMNadenylyl transferase (that releases pyrophosphate). The most important thing you have to be aware of is cross contamination of your reaction with free phosphate. This is generally observed in most phosphatase reactions. Make sure both your enzyme of interest as well as the substrate does not contain free Pi to begin with.

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A generic Phosphatase and pyrophosphatase assay